Cardiovascular diseases account for 40% of death in United States. Vascular diseases such as atherosclerosis and restenosis involve extensive vascular remodeling such as cell proliferation and migration in neointima. Vascular smooth muscle cells (SMCs) are a major cell type in the tunica media of vascular wall, and it is generally accepted that mature SMCs can de-differentiate into proliferative/synthetic phenotype during vascular remodeling. However, there is a lack of direct evidence that characterize the de-differentiation of mature SMCs in vitro and in vivo, and the relative contribution of SMCs and vascular stem cells to disease development has not been well elucidated. Here we aim to address this knowledge gap. Recently, we have identified a novel type of multipotent vascular stem cells (MVSCs) in the wall of blood vessels. MVSCs express transcriptional markers such as Sox10 and Sox17, and can differentiate into both proliferative/synthetic and mature SMCs. In addition, MVSCs can differentiate into several mesenchymal (chondrogenic, adipogenic and osteogenic) and neural (peripheral neuron and Schwann cell) lineages. By using DNA microarray analysis, we have also identified a potential MVSC surface marker that may regulate MVSC proliferation and migration. Furthermore, by using lineage tracing and biochemical analysis, we have shown that MVSCs outgrow SMCs in culture and that MVSCs become proliferative following vascular injury and contribute to neointima formation. Finally, we have isolated and characterized MVSCs from human atherosclerotic plaques, coronary artery, aorta and femoral artery. Based on our preliminary studies, we hypothesize that MVSC differentiation is a major source of proliferative/synthetic SMCs and contributes to neointima formation. To test our hypothesis, three Specific Aims are proposed: (1) Characterize MVSC markers and the relative contribution of MVSCs and mature SMCs to proliferative/synthetic SMCs in vitro by using lineage tracing models; (2) Determine the roles of MVSCs and mature SMCs in neointima formation following vascular injury in transgenic mouse models; (3) Characterize MVSCs in human arteries and atherosclerotic lesions. If accomplished, this work will break new grounds in vascular biology, and demonstrate the important role of MVSC differentiation in vascular remodeling and disease development. This work will also provide insight into the mechanisms of vascular remodeling and lead to the development of novel therapies for vascular diseases by using MVSC as a therapeutic target.